The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS), an ultimately lethal condition. The virus currently infects 35 million people worldwide and in 2013 alone, AIDS caused 1.5 million deaths. A key feature of the relationship between HIV and AIDS is that the viral infection itself is not lethal, but instead the virus indirectly kills the patient through the progressive destruction of the immune system leading to AIDS. Thus, a functional cure would entail creating an immune system that is resistant to destruction by HIV. Replacement of the hematopoietic system with cells engineered to confer HIV resistance might also lead to a sterilizing cure (the complete abolition of the virus from the patient) through the virus eliminating its susceptible sanctuary cells and having them replaced by engineered resistant cells over time. Toward this, others have focused on using engineered nuclease mediated genome editing by non-homologous end-joining to mutate and disrupt expression of the CCR5 co-receptor gene as a genetic approach to blocking initial HIV entry into cells. Our focus instead is to use homologous recombination mediated genome editing to knock-in a cocktail of anti-HIV genes at the CCR5 locus thereby simultaneously expressing multiple anti-HIV genetic protective factors and disrupting the CCR5 gene-a form of genetic cART. By engineering cells to have a new property we are combining the principles of genome editing with synthetic biology. In this proposal we focus on establishing a highly effective and non-cytotoxic cocktail of anti-HIV genes (an anti-HIV operon) and determining the best and safest nuclease platform to target the insertion of this anti-HIV operon into the CCR5 gene in CD34+ hematopoietic stem and progenitor cells (HSPCs). Our hypothesis is that we will effectively and safely generate an HIV resistant immune system (including T-cells and macrophages) that will provide a stable long-term genetic cure to HIV. To achieve this goal we have three specific aims. Aim 1 will determine which combination of anti-HIV genes are the most effective and do not perturb the normal functioning of the modified cells. Aim 2 will determine the effectiveness and safety of using CCR5-TALEN mediated genome editing in CD34+ HSPCS and compare this to the developed CCR5-ZFNs. Aim 3 will determine the effectiveness and safety of using CCR5-CRISPR mediated genome editing in CD34+ HSPCS and compare this to the developed CCR5-ZFNs. Collectively these aims will identify the most effective anti-HIV operon and the optimal nuclease delivery platform for generation of stably engineered HIV resistant cells.